Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

Rosa R. Jersie-Christensen, Abida Sultan, Jesper V Olsen, Louise von Stechow (Editor)

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissociation (HCD).
    Original languageEnglish
    JournalMethods in Molecular Biology
    Volume1355
    Pages (from-to)251-260
    Number of pages10
    ISSN1064-3745
    DOIs
    Publication statusPublished - 2016

    Keywords

    • Heated guanidinium chloride lysis
    • Mass spectrometry
    • Phosphopeptide enrich ment
    • Phosphoproteomics
    • Sample preparation

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